pain-enhancing mechanism through interaction between trpv1 ... · teraction of mtrpv1 with mano1...

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Pain-enhancing mechanism through interaction between TRPV1 and anoctamin 1 in sensory neurons Yasunori Takayama a , Daisuke Uta b , Hidemasa Furue c,d , and Makoto Tominaga a,d,1 a Division of Cell Signaling, Okazaki Institute for Integrative Bioscience, Okazaki 444-8787, Japan; b Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan; c Division of Neural Signaling, National Institute for Physiological Sciences, Okazaki 444-8787, Japan; and d Department of Physiological Sciences, Graduate University for Advanced Studies, Okazaki 444-8787, Japan Edited by David Julius, University of California, San Francisco, CA, and approved March 20, 2015 (received for review November 11, 2014) The capsaicin receptor transient receptor potential cation channel vanilloid 1 (TRPV1) is activated by various noxious stimuli, and the stimuli are converted into electrical signals in primary sensory neurons. It is believed that cation influx through TRPV1 causes depolarization, leading to the activation of voltage-gated sodium channels, followed by the generation of action potential. Here we report that the capsaicin-evoked action potential could be induced by two components: a cation influx-mediated depolarization caused by TRPV1 activation and a subsequent anion efflux-mediated de- polarization via activation of anoctamin 1 (ANO1), a calcium-activated chloride channel, resulting from the entry of calcium through TRPV1. The interaction between TRPV1 and ANO1 is based on their physical binding. Capsaicin activated the chloride currents in an extracellular calcium-dependent manner in HEK293T cells expressing TRPV1 and ANO1. Similarly, in mouse dorsal root ganglion neurons, capsaicin- activated inward currents were inhibited significantly by a specific ANO1 antagonist, T16Ainh-A01 (A01), in the presence of a high concentration of EGTA but not in the presence of BAPTA [1,2-bis (o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid]. The genera- tion of a capsaicin-evoked action potential also was inhibited by A01. Furthermore, pain-related behaviors in mice treated with cap- saicin, but not with αβ-methylene ATP, were reduced significantly by the concomitant administration of A01. These results indicate that TRPV1ANO1 interaction is a significant pain-enhancing mecha- nism in the peripheral nervous system. pain perception | primary sensory neuron | anoctamin 1 | TRPV1 W hen calcium ions enter cells through ion channels or trans- porters, they can initiate a variety of reactions, either as free calcium ions or after their binding by specific calcium-binding proteins (1). One such important reaction is the activation of calcium-binding proteins by calcium nanodomains of the calcium pathways (2). In this regard, some transient receptor potential (TRP) channels have high calcium permeability (3), and it is likely that they activate calcium-binding proteins in the cytosol or plasma membrane. Indeed, TRP vanilloid 4 (TRPV4), a thermo- sensitive TRP channel (reportedly an osmo- or mechano-sensor) (48) and anoctamin 1 (ANO1; a calcium-activated chloride channel) (911) function as a complex in epithelial cells of the choroid plexus (12). Upon entering choroid plexus epithelial cells, calcium activates ANO1, leading to chloride efflux. Al- though the interaction between TRP channels and anoctamins could work in a variety of ways (12), the direction of chloride movement is determined simply by the relationship between chloride equilibrium potentials and membrane potentials, depend- ing on the intracellular chloride concentrations (13). This concept prompted us to pursue other interactions between TRP channels and anoctamins. We focused on primary sensory neurons because activation of chloride channels in sensory neurons causes chloride efflux and depolarization, as the result of the high intracellular chloride concentrations (14, 15). TRPV1 senses various noxious stimuli in primary sensory neurons, leading to pain perception through the generation of action potentials upon the activation of voltage-gated sodium channels. Mammalian TRPV1 is activated by noxious heat, acid, and many chemical compounds including capsaicin (1618). The calcium permeability of TRPV1 is more than 10 times that of sodium, suggesting that TRPV1 could activate anoctamins readily, leading to further depolarization. ANO1 plays an important role in noci- ception in primary sensory neurons (19), and bradykinin-induced and neuropathic pain-related behaviors were reduced in ANO1 conditional-knockout mice (20, 21), suggesting that interaction be- tween the two proteins could strongly enhance nociceptive signals. Results Coexpression of TRPV1 and ANO1 in Dorsal Root Ganglions and Their Interaction in HEK293T Cells. We first examined the coexpression of TRPV1 and ANO1 in mouse dorsal root ganglion (DRG) neu- rons and found that they indeed were coexpressed as previously reported (19). Specifically, 78.2% (136/174 TRPV1-expressing neurons) of anti-TRPV1 antibody-immunoreactive neurons also reacted with an anti-ANO1 antibody (Fig. 1 AC). Next, we in- vestigated the interaction between TRPV1 and ANO1. We used a whole-cell patch-clamp technique in HEK293T cells expressing mouse TRPV1 (mTRPV1) and mouse ANO1 (mANO1) or cells expressing either mTRPV1 or mANO1 alone. In these experi- ments, we used an N-methyl-D-glucamine chloride (NMDG-Cl) bath and pipette solutions that allowed us to observe chloride currents (Fig. 1 DG). Capsaicin-activated chloride currents with a linear currentvoltage relationship were significantly greater in cells expressing mTRPV1 and mANO1 than in cells expressing either mTRPV1 or mANO1 alone, depending on the extracellular calcium concentrations (Fig. 1 DF). These results suggested that mANO1 is activated by calcium influx through mTRPV1 activation. The finding that there was no outward current activation with an Significance The capsaicin receptor transient receptor potential cation channel vanilloid 1 (TRPV1) and anoctamin 1 (ANO1) work as receptors activated by noxious stimuli in sensory nerve endings. It is be- lieved that activation of the two channels causes cation influx and anion efflux, respectively, both of which lead to de- polarization. We show that ANO1 is activated by calcium ions entering neurons through TRPV1 activation based on their physical binding on the cell membrane. Indeed, both capsaicin- activated inward currents in sensory neurons and capsaicin- induced pain-related behaviors in mice were inhibited signifi- cantly by ANO1 blockade. To our knowledge, this is the first evidence for a mechanism by which interaction between TRPV1 and ANO1 functions as a pain-enhancing mechanism. Author contributions: Y.T., D.U., H.F., and M.T. designed research; Y.T. and D.U. per- formed research; Y.T. and D.U. analyzed data; and Y.T., H.F., and M.T. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1 To whom correspondence should be addressed. Email: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1421507112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1421507112 PNAS | April 21, 2015 | vol. 112 | no. 16 | 52135218 NEUROSCIENCE Downloaded by guest on November 21, 2020

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Page 1: Pain-enhancing mechanism through interaction between TRPV1 ... · teraction of mTRPV1 with mANO1 was detected by immuno-precipitation of cells expressing mTRPV1 and mANO1 but not

Pain-enhancing mechanism through interactionbetween TRPV1 and anoctamin 1 in sensory neuronsYasunori Takayamaa, Daisuke Utab, Hidemasa Furuec,d, and Makoto Tominagaa,d,1

aDivision of Cell Signaling, Okazaki Institute for Integrative Bioscience, Okazaki 444-8787, Japan; bDepartment of Applied Pharmacology, Graduate Schoolof Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan; cDivision of Neural Signaling, National Institute for PhysiologicalSciences, Okazaki 444-8787, Japan; and dDepartment of Physiological Sciences, Graduate University for Advanced Studies, Okazaki 444-8787, Japan

Edited by David Julius, University of California, San Francisco, CA, and approved March 20, 2015 (received for review November 11, 2014)

The capsaicin receptor transient receptor potential cation channelvanilloid 1 (TRPV1) is activated by various noxious stimuli, and thestimuli are converted into electrical signals in primary sensoryneurons. It is believed that cation influx through TRPV1 causesdepolarization, leading to the activation of voltage-gated sodiumchannels, followed by the generation of action potential. Here wereport that the capsaicin-evoked action potential could be inducedby two components: a cation influx-mediated depolarization causedby TRPV1 activation and a subsequent anion efflux-mediated de-polarization via activation of anoctamin 1 (ANO1), a calcium-activatedchloride channel, resulting from the entry of calcium through TRPV1.The interaction between TRPV1 and ANO1 is based on their physicalbinding. Capsaicin activated the chloride currents in an extracellularcalcium-dependent manner in HEK293T cells expressing TRPV1 andANO1. Similarly, in mouse dorsal root ganglion neurons, capsaicin-activated inward currents were inhibited significantly by a specificANO1 antagonist, T16Ainh-A01 (A01), in the presence of a highconcentration of EGTA but not in the presence of BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid]. The genera-tion of a capsaicin-evoked action potential also was inhibited byA01. Furthermore, pain-related behaviors in mice treated with cap-saicin, but not with αβ-methylene ATP, were reduced significantlyby the concomitant administration of A01. These results indicatethat TRPV1–ANO1 interaction is a significant pain-enhancing mecha-nism in the peripheral nervous system.

pain perception | primary sensory neuron | anoctamin 1 | TRPV1

When calcium ions enter cells through ion channels or trans-porters, they can initiate a variety of reactions, either as free

calcium ions or after their binding by specific calcium-bindingproteins (1). One such important reaction is the activation ofcalcium-binding proteins by calcium nanodomains of the calciumpathways (2). In this regard, some transient receptor potential(TRP) channels have high calcium permeability (3), and it islikely that they activate calcium-binding proteins in the cytosol orplasma membrane. Indeed, TRP vanilloid 4 (TRPV4), a thermo-sensitive TRP channel (reportedly an osmo- or mechano-sensor)(4–8) and anoctamin 1 (ANO1; a calcium-activated chloridechannel) (9–11) function as a complex in epithelial cells of thechoroid plexus (12). Upon entering choroid plexus epithelialcells, calcium activates ANO1, leading to chloride efflux. Al-though the interaction between TRP channels and anoctaminscould work in a variety of ways (12), the direction of chloridemovement is determined simply by the relationship betweenchloride equilibrium potentials and membrane potentials, depend-ing on the intracellular chloride concentrations (13). This conceptprompted us to pursue other interactions between TRP channelsand anoctamins. We focused on primary sensory neurons becauseactivation of chloride channels in sensory neurons causes chlorideefflux and depolarization, as the result of the high intracellularchloride concentrations (14, 15).TRPV1 senses various noxious stimuli in primary sensory

neurons, leading to pain perception through the generation ofaction potentials upon the activation of voltage-gated sodium

channels. Mammalian TRPV1 is activated by noxious heat, acid, andmany chemical compounds including capsaicin (16–18). The calciumpermeability of TRPV1 is more than 10 times that of sodium,suggesting that TRPV1 could activate anoctamins readily, leadingto further depolarization. ANO1 plays an important role in noci-ception in primary sensory neurons (19), and bradykinin-inducedand neuropathic pain-related behaviors were reduced in ANO1conditional-knockout mice (20, 21), suggesting that interaction be-tween the two proteins could strongly enhance nociceptive signals.

ResultsCoexpression of TRPV1 and ANO1 in Dorsal Root Ganglions and TheirInteraction in HEK293T Cells.We first examined the coexpression ofTRPV1 and ANO1 in mouse dorsal root ganglion (DRG) neu-rons and found that they indeed were coexpressed as previouslyreported (19). Specifically, 78.2% (136/174 TRPV1-expressingneurons) of anti-TRPV1 antibody-immunoreactive neurons alsoreacted with an anti-ANO1 antibody (Fig. 1 A–C). Next, we in-vestigated the interaction between TRPV1 and ANO1. We useda whole-cell patch-clamp technique in HEK293T cells expressingmouse TRPV1 (mTRPV1) and mouse ANO1 (mANO1) or cellsexpressing either mTRPV1 or mANO1 alone. In these experi-ments, we used an N-methyl-D-glucamine chloride (NMDG-Cl)bath and pipette solutions that allowed us to observe chloridecurrents (Fig. 1 D–G). Capsaicin-activated chloride currents witha linear current–voltage relationship were significantly greater incells expressing mTRPV1 and mANO1 than in cells expressingeither mTRPV1 or mANO1 alone, depending on the extracellularcalcium concentrations (Fig. 1 D–F). These results suggested thatmANO1 is activated by calcium influx through mTRPV1 activation.The finding that there was no outward current activation with an

Significance

The capsaicin receptor transient receptor potential cation channelvanilloid 1 (TRPV1) and anoctamin 1 (ANO1) work as receptorsactivated by noxious stimuli in sensory nerve endings. It is be-lieved that activation of the two channels causes cation influxand anion efflux, respectively, both of which lead to de-polarization. We show that ANO1 is activated by calcium ionsentering neurons through TRPV1 activation based on theirphysical binding on the cell membrane. Indeed, both capsaicin-activated inward currents in sensory neurons and capsaicin-induced pain-related behaviors in mice were inhibited signifi-cantly by ANO1 blockade. To our knowledge, this is the firstevidence for a mechanism by which interaction between TRPV1and ANO1 functions as a pain-enhancing mechanism.

Author contributions: Y.T., D.U., H.F., and M.T. designed research; Y.T. and D.U. per-formed research; Y.T. and D.U. analyzed data; and Y.T., H.F., and M.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1421507112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1421507112 PNAS | April 21, 2015 | vol. 112 | no. 16 | 5213–5218

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extracellular NMDG-aspartate and an intracellular NMDG-Cl so-lution (Fig. 1G, red) further indicated that chloride was the chargecarrier of the observed currents. Furthermore, the physical in-teraction of mTRPV1 with mANO1 was detected by immuno-precipitation of cells expressing mTRPV1 and mANO1 but notcells expressing either mTRPV1 or mANO1 alone (Fig. 1H).These results indicated that the functional interaction is basedon physical binding.TRP subfamily A, member 1 (TRPA1), another nociceptive TRP

channel with high calcium permeability, and TRPV1 are coex-pressed in some DRG neurons, and they reportedly interact witheach other as a functional complex (22, 23). Therefore, we ex-amined TRPA1–ANO1 interaction using a whole-cell patch-clamp technique in HEK293T cells expressing mTRPA1 andmANO1. We found their functional interaction depended onthe extracellular calcium concentrations, observations similar tothose made with mTRPV1 and mANO1 (Fig. S1 A–C). Althoughsome reports have shown an interaction between TRPV1 andTRPA1 (22, 23), capsaicin-activated chloride currents did notdiffer between cells expressing mTRPV1 and mANO1 and cellsexpressing mTRPV1, mTRPA1, and mANO1, suggesting thatinteraction between mTRPV1 and mANO1 is not affected by theTRPV1–TRPA1 interaction (Fig. S1D).

TRPV1–ANO1 Interaction in DRG Neurons. Next, we investigatedthe proteins’ interaction in small (<24 μm in diameter) DRGneurons by observing capsaicin-activated currents using an NaCl

bath (containing 2 mM calcium) and an NMDG-Cl pipette so-lution in which sodium inward, calcium inward, and chlorideoutward movements were observed as inward currents. Thus,capsaicin-activated inward currents could include both TRPV1-mediated cation currents and ANO1-mediated anion currents.Approximately 80% of TRPV1-positive neurons expressed ANO1(Fig. 1), indicating that the TRPV1–ANO1 interaction was stronglyinvolved in the reactions in native neurons. As expected, capsaicin-activated currents were reduced by an ANO1 inhibitor, T16Ainh-A01 (A01), in small DRG neurons with 0.2 and 5 mM EGTA(Fig. 2A). However, current reductions were not observed usinga faster chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA; 5 mM), suggesting that the TRPV1–ANO1 interaction occurred in a local calcium nanodomain (24).Furthermore, the observation that A01 did not affect the inwardcurrents without extracellular calcium in HEK293T cells expressingmTRPV1 alone (Fig. 2B) argued against off-target effects of A01in TRPV1 activation by capsaicin. The direct interaction be-tween TRPV1 and ANO1 in DRGs (Fig. 2C) supported our ideathat calcium ions entering the cells through TRPV1 immediatelyactivated ANO1, consistent with the data obtained with calciumchelators. We also analyzed A01 effects in time courses of thenormalized currents, but they were not changed significantly inDRG neurons regardless of chelator concentrations (Fig. S2).These results suggested that A01 affected only TRPV1-mediatedevents without modulating its kinetics in DRG neurons, although

Fig. 1. Coexpression of TRPV1 and ANO1 in the DRG and TRPV1–ANO1 interaction in HEK293T cells. (A and B) Immunofluorescent images with an anti-TRPV1antibody (green) and an anti-ANO1 antibody (red) in 10-μm DRG slices from mice. Images at the far right in A and B are bright-field images. (Scale bars,50 μm.) (C) Cell size-dependent distribution of mouse DRG neurons expressing both TRPV1 and ANO1, TRPV1 alone, or ANO1 alone (n = 444 cells).(D) Representative traces of whole-cell chloride currents activated by capsaicin in HEK293T cells expressing mTRPV1 and mANO1 (Top), cells expressingmTRPV1 alone (Middle), and mock-transfected cells (Bottom). Holding potential was −60 mV, and ramp pulses were from −100 to +100 mV. (Inset) Current–voltage (I–V) curves at the time indicated by black and red arrows in D, Top. (E and F) Comparison of the peak currents in cells expressing mTRPV1 andmANO1, cells expressing mTRPV1 alone, and cells expressing mANO1 alone (E ; n = 5 cells) and the effect of extracellular calcium (F ; n = 5 cells). Data are shownas the mean ± SEM of five peaks. **P < 0.01; *P < 0.05. (G) I–V relationship of capsaicin-activated currents with NMDG-Cl (black; n = 4 cells) and NMDG-aspartate (red; n = 3 cells) extracellular solutions. (H) Physical interaction between TRPV1 and ANO1 in HEK293T cells. Samples were immunoprecipitated (IP)with an anti-ANO1 antibody and blotted with an anti-TRPV1 antibody.

5214 | www.pnas.org/cgi/doi/10.1073/pnas.1421507112 Takayama et al.

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the capsaicin-activated current was prolonged in HEK293T cellsexpressing TRPV1 and ANO1 (Fig. 1D).Thus, TRPV1 activation appeared to have dual effects on

membrane potentials: a cation influx-evoked depolarization anda chloride efflux-evoked depolarization. Next, to investigatewhether TRPV1–ANO1 interaction is involved in generatingaction potential, we observed capsaicin-evoked action potentialsin isolated small DRG neurons with or without A01. Thecurrent-clamp recordings were performed under conditions inwhich the calculated equilibrium chloride potential was approxi-mately −20 mV, which is within the physiological range (15). ATP(4 mM) was included in the potassium-base pipette solution tomaintain the activation of the sodium–potassium pump andTRPV1 (25). The action potential generated by the secondcapsaicin application disappeared with 10 μM A01 in 16 of 18neurons that responded to the first capsaicin application (Fig. 2D, F, and G). We confirmed that the neurons still had the abilityto show action potentials after the second capsaicin applicationwith a depolarizing current injection (Fig. 2D, Far Right). Incontrast, action potential generation was observed in 10 of 18DRG neurons in the second capsaicin application without A01(Fig. 2 E and F), and the difference in the action potentialnumbers was statistically significant (Fig. 2G). Moreover, therelative number of action potentials was reduced drastically inthe presence of A01 (Fig. 2H). We also checked whether A01affected the voltage-gated channels. The results showed that A01did not change voltage-gated sodium (Nav), potassium (Kv), orcalcium (Cav, with barium as a charge carrier) channel currents,but Nav, Kv, and Cav channel currents were reduced drasticallyin the NMDG-Cl bath solution, in the bath solution with

tetraethylammonium (TEA), and in the barium-free bath solu-tion, respectively (Fig. 2 I–K). Furthermore, the action potentialsevoked by depolarizing current injection were not affected byA01 (Fig. 2L). These results indicated that TRPV1–ANO1 in-teraction could affect neural excitation in small DRG neurons.

Effects of an ANO1 Blocker in Capsaicin-Induced Pain-Related Behaviors.The data presented thus far suggested that inhibition of ANO1might reduce capsaicin-induced pain-related behaviors. Therefore,we investigated the licking behaviors of mice whose hind pawswere injected with 10 μL of capsaicin (300 μM) with or withoutA01 (300 μM). As expected, capsaicin-induced pain-related be-haviors for the first 30 s and for a total of 5 min were reducedsignificantly by concomitant administration of A01, whereas lickingbehaviors induced by αβ-methylene ATP were not affected by A01,and A01 itself did not cause any pain-related behavior (Fig. 3 A–F).These data indicated that the effects of A01 were specific for thecapsaicin-induced pain-related behaviors, supporting a specific in-teraction between TRPV1 and ANO1.

TRPV1–ANO1 Interaction in Presynaptic Terminals. Proteins pro-duced in DRG cell bodies are transported both to sensory nerveendings and to presynaptic termini of primary afferent neuronsin the spinal cord (17), suggesting that TRPV1–ANO1 inter-action in the presynaptic termini could be involved in synaptictransmission. Therefore, we compared the capsaicin-induced fa-cilitation of spontaneous excitatory postsynaptic currents (sEPSCs)in substantia gelatinosa (SG) neurons of the superficial spinaldorsal horn of mice. Six of 19 neurons (31.5%) did not respondto capsaicin administration to the spinal cord preparation (Fig. S3),

Fig. 2. TRPV1–ANO1 interaction in DRG neurons. (A) Capsaicin-activated currents at −60 mV (including 0.2 mM EGTA, 5 mM EGTA, or 5 mM BAPTA) without(n = 24, 13, and 10 cells, respectively) or with (n = 20, 11, and 9 cells, respectively) an ANO1 blocker, T16Ainh-A01 (A01), in the presence of extracellularcalcium in DRG neurons. (B) Capsaicin-activated currents in HEK293T cells expressing mTRPV1 alone in the absence of extracellular calcium (n = 5 cells). (C)Physical interaction between TRPV1 and ANO1 in DRG. (D) A representative trace of the capsaicin-evoked action potential of a DRG neuron. A depolarizingcurrent was injected to check the action potential generation. (E) A representative trace of the capsaicin-evoked action potentials without A01. (F and G)Comparison of the action potential numbers in each cell before and after A01 application (n = 18 cells each). (H) Comparison of the relative number of actionpotentials (n = 18 cells each). (I –K) Current–voltage relationships of the voltage-gated channel currents with (red) or without (black) A01. Blue lines show theloss of Nav (I), Kv (J), and Cav (K) channel currents (n = 4–9 cells). The holding potential was −60 mV, and step pulses were applied. (L) Averaged frequency ofaction potentials for 3 s evoked by depolarizing current injection (+150 pA) with or without A01 (n = 8 cells each). Basal voltage was kept at −60 mV. Data areshown as mean ± SEM; *P < 0.05. n.s., not significant.

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a ratio similar to a previous report (26). Among the responsive13 neurons, we compared the facilitation of sEPSCs upon thesecond capsaicin application after a long interval that shouldhave minimized the desensitization through calmodulin binding.Facilitation of sEPSC frequencies was observed in the first twocapsaicin applications in the absence of A01 (Fig. 4 A–C). Suchfacilitation was reduced significantly, by approximately one-half, in the presence of A01, although the amplitudes wereunchanged (Fig. 4 D–F). These results suggested that A01inhibits glutamate release through the interaction betweenTRPV1 and ANO1 without postsynaptic effects.To prove further that the effects of ANO1 on sEPSCs are

specific to TRPV1-mediated events without the involvement ofother calcium-permeable channels, we examined the effects ofA01 on the root evoked EPSCs (eEPSCs) by electrical stimula-tion. We did not observe any effect of A01 on the eEPSCs in theSG neurons with monosynaptic Aδ- or C-fiber input (Fig. 4G),suggesting specific ANO1 involvement upon TRPV1 activation.

DiscussionWe found that the capsaicin-evoked depolarization was com-posed of two components: a cation influx-mediated depolariza-tion caused by TRPV1 activation and a subsequent anion efflux-mediated depolarization via ANO1 activation caused by the entry ofcalcium through TRPV1. The physical binding of TRPV1 to ANO1could facilitate their interaction, and calcium within the nano-domains of the TRPV1–ANO1 complex could participate in theinteraction, as is consistent with the observation that EGTA is to-tally ineffective on free calcium within 20 nm of a channel (24). Thegeneration of action potential through TRPV1 activation wasinhibited by an ANO1 blocker, A01 (Fig. 2 D–H). The reduction inaction potential generation also was observed in the absence ofA01, although less frequently, partly because TRPV1 could bedesensitized to some extent even in the presence of ATP and be-cause TRPV1 activation induces the inactivation of Nav channels(27). Nevertheless, these results indicate that the TRPV1–ANO1interaction could induce the generation of action potential in DRGneurons. Moreover, the facilitated action potentials could lead toenhanced glutamate release at the synapse in the spinal cord (Fig.3G), followed by more pain-related behaviors (Fig. 3 A and B). Inthe presynaptic terminals, TRPV1 is not exposed to capsaicin orhigh temperatures but could be activated by reported endogenousactivators (28) or body temperature because temperature thresholdsfor TRPV1 activation reportedly are reduced to the body-temper-ature range under inflammatory conditions (29). TRPV1–ANO1interaction in the spinal cord could enhance synaptic transmissionunder conditions in which it could be inhibited by TRPV1–ANO1interaction blockade (Fig. 4H). Moreover, A01 effects were notobserved in root-evoked EPSCs (Fig. 4G) although intracellularcalcium ions should be increased locally in the presynaptic ter-minals. Thus, TRPV1–ANO1 interaction should occur with localcalcium increases within nanodomains, although it is possiblethat global calcium increases contribute to ANO1 activation tosome extent.TRPV1 and TRPA1 have high calcium permeability compared

with other channels involved in nociception, e.g., P2X receptors(30). Influxes of calcium and sodium upon activation of TRPV1cause depolarization, leading to the generation of the actionpotential. Furthermore, increased intracellular calcium leads tothe release of several peptides, including CGRP and substance P, aphenomenon known as “neurogenic inflammation” (31). Thus, we

Fig. 3. Reduction of capsaicin-evoked pain-related behaviors by A01 and aschematic model for TRPV1–ANO1 interaction in pain sensation. (A and B)Time courses of the capsaicin-induced pain-related licking behaviors with(n = 7 mice) or without (n = 8 mice) A01 every 30 s (A) and the summation for5 min (B). Both capsaicin and A01 concentrations were 300 μM (10 μL). Dataare shown as mean ± SEM; *P < 0.05. (C and D) Time courses of αβ-methyleneATP (αβmeATP)-induced pain-related licking behaviors with (n = 7 mice) orwithout (n = 8 mice) A01 every 30 s (C) and the summation over 5 min (D).αβmeATP and A01 concentrations were 10 mM and 300 μM, respectively(10 μL). Data are shown as mean ± SEM. (E and F) Time courses of lickingbehaviors with (n = 7 mice) or without (n = 7 mice) A01 every 30 s (E) and thesummation over 5 min (F). A01 concentration was 300 μM (10 μL). Data areshown as mean ± SEM. (G) A schematic model based on the data obtainedshowing TRPV1–ANO1 interaction in pain sensation. Capsaicin-evokeddepolarization (Δψ) contains two components: a cation influx-mediateddepolarization through TRPV1 activation and a chloride efflux-mediated

depolarization through ANO1 activation caused by calcium entering the cellsthrough TRPV1. Summation could lead to the generation of an enhancedaction potential (Upper), followed by increased glutamate release (Lower).Inhibition of ANO1 or the TRPV1-ANO1 interaction could reduce the gluta-mate release (Lower).

5216 | www.pnas.org/cgi/doi/10.1073/pnas.1421507112 Takayama et al.

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propose an additional role for TRPV1’s high calcium permeability:That the activation of ANO1 bound to TRPV1 produces furtherdepolarization through chloride efflux.Our results indicate that a significant portion of capsaicin-

evoked events involve TRPV1–ANO1 interaction. Many TRPV1antagonists have been used to reduce pain in clinical trials. How-ever, most have not shown efficacy, and patients have noted hy-perthermia and abnormal thermal sensations as side effects (32, 33).Therefore, inhibiting the TRPV1–ANO1 interaction could be analternative way to treat sensations of pain. In acute inflammatorypain, TRPV1 is sensitized by protein kinase C downstream from Gqprotein-coupled receptors, including an ATP receptor, thereby re-ducing the temperature threshold for TRPV1 activation (29, 34). Inneuropathic pain models, TRPV1 function and expression re-portedly are increased (35). Therefore, it is expected that ANO1activation caused by TRPV1 activity also should be enhanced underthose conditions. Furthermore, it has been reported recently thatANO1 is involved in neuropathic pain in mice (21). All these factsclearly suggest that the inhibition of TRPV1–ANO1 interactionwould be an intriguing way to develop novel analgesic agents.

Materials and MethodsAnimals. All mouse experiments were conducted with 6- to 8-wk-old C57BL/6NCr mice. All animal experimental procedures were performed accordingto the National Institute for Physiological Sciences guidelines.

Whole-Cell Voltage-Clamp Recordings in HEK293T Cells. HEK293T cells werecultured in DMEM (high glucose) with L-glutamine and phenol red (Wako)containing 10% (vol/vol) FBS (lot no. S06537S1560; Biowest), penicillin/streptomycin (1:200; Life Technologies), and GlutaMAX (1:100; Life Tech-nologies) at 37 °C in humidified air containing 5% CO2. The cells were

transfected with cDNA carrying mouse Trpv1, mouse Trpa1, mouse Ano1 (agenerous gift from U. Oh, Seoul National University, Seoul, Korea), orpcDNA3.1 using Lipofectamine (Invitrogen). The cells were used 24–36 hafter transfection. The bath solution contained 140 mM NMDG-Cl or NMDG-aspartate, 1 mM MgCl2, 2 mM CaCl2, or 5 mM EGTA, 10 mM glucose, and10 mM Hepes, pH 7.40. The pipette solution contained 140 mM NMDG-Cl orKCl, 1 mMMgCl2, 5 mM BAPTA, and 10 mM Hepes, pH 7.30. The free calciumconcentration of the pipette solution was maintained at 100 nM calculatedwith the MAXC program (Stanford University). The holding potential was−60 mV, and ramp pulses from −100 to +100 mV were applied for 300 msevery 5 s. Currents were recorded using an Axopatch 200B amplifier (MolecularDevices), filtered at 5 kHz with a low-pass filter, and digitized with Digidata1440A (Axon Instruments). pCLAMP 10 (Axon Instruments) data acquisitionsoftware, was used.

Whole-Cell Voltage-Clamp Recordings in DRG Neurons. Data were collectedfrom small (<24 μm) DRG neurons using the setup described above forwhole-cell voltage-clamp recordings of HEK293T cells. The basic NaCl bathsolution contained 130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2,10 mM glucose, and 10 mM Hepes, pH 7.40. The pipette solution (pH 7.30)contained 140 mM NMDG-Cl, 1 mMMgCl2, 10 mM Hepes with 0.2 mM EGTA,5 mM EGTA, or 5 mM BAPTA. When we recorded currents of Kv and Cavchannels, we changed the composition of the bath solution from NaCl toNMDG-Cl. We replaced KCl with CsCl for the recordings of Nav channelcurrents and with NMDG-Cl for recordings of Cav channel currents. EGTA(5 mM) was added to all bath solutions for the recordings of Nav, Kv, andCav channel currents. We used BaCl2 (the free concentration was 2 mMcalculated using the MAXC program) instead of CaCl2 for recordings of Cavchannel currents. The basic KCl pipette solution contained 140 mM KCl;1 mM MgCl2; 5 mM EGTA or BAPTA or 0.2 mM EGTA; and 10 mM Hepes,pH 7.30. We changed the composition of the pipette solution from KCl to CsClor NMDG-Cl for the recordings of Nav or Cav channel currents, respectively.BAPTA (5 mM) was added to the pipette solutions for the recordings of Cavchannel currents, and 0.2 mM EGTA was added to the pipette solutions for

Fig. 4. The effects of A01 on sEPSCs and eEPSCs and a schematic model for TRPV1-ANO1 interaction in the spinal cord of mice. (A) A representative trace(Upper) with magnified traces (Lower) of sEPSCs obtained in an SG neuron without A01. The holding potential was −70 mV. (B and C) Comparison of thefrequencies (B) and amplitudes (C) of sEPSCs before and after the first and second capsaicin applications (n = 8 cells). (D) A representative trace (Upper) withmagnified traces (Lower) of sEPSCs with A01 (10 μM). (E and F) Comparison of the frequencies (E) and amplitudes (F) of sEPSCs before and after the first andsecond capsaicin applications (n = 5 cells). Data are shown as mean ± SEM; **P < 0.01; n.s., not significant. (G, Upper) Representative root eEPSCs in the SGneurons with monosynaptic Aδ- (Left) or C- (Right) fiber input in the absence (black) and presence (red) of A01 (10 μM). stim, electrical stimulation. (G, Lower)Comparison of the A01 effects with and without A01 in Aδ- (Left; n = 6 cells) and C-fiber (Right; n = 7 cells). (H) A schematic model showing that glutamatereleases through the TRPV1–ANO1 interaction in a presynaptic terminus. (Left) Certain endogenous agonists and elevated body temperature during in-flammation could activate TRPV1, and TRPV1–ANO1 interaction could facilitate depolarization, leading to further activation of the Cav channels, followed byincreased glutamate release. (Right) Inhibition of the TRPV1–ANO1 interaction could lead to reduced glutamate release.

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Page 6: Pain-enhancing mechanism through interaction between TRPV1 ... · teraction of mTRPV1 with mANO1 was detected by immuno-precipitation of cells expressing mTRPV1 and mANO1 but not

the recordings of capsaicin-activated currents in DRG neurons. The freecalcium concentration of the pipette solution was maintained at 100 nMexcept for the recordings of voltage-gated channel currents. The holdingpotential was −60 mV, and step pulses from −100 to +100 mV (Δ20 mV) wereapplied for 100–500 ms every 3 s.

Whole-Cell Current-Clamp Recordings of DRG Neurons. Whole-cell current-clamp recordings of DRG neurons were collected in the setup used for whole-cell voltage-clamp recordings. The bath solution contained 130 mM NaCl,5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, and 10 mM Hepes,pH 7.40, with NaOH. The pipette solution contained 67 mM KCl, 65 mMK-gluconate, 1 mM MgCl2, 5 mM EGTA, 4 mM ATP-Mg, 1 mM GTP-Na2, and10 mM Hepes, pH 7.30, with KOH. Free calcium was maintained at 100 nM.Basal voltages were maintained around −60 mV by current injection.

Whole-Cell Patch-Clamp Recordings from Dorsal Horn Neurons in the SpinalCord. The SG was easily discernible with transmitted illumination as a rela-tively translucent band across the dorsal horn in the transverse slice prep-arations. Blind whole-cell voltage-clamp recordings were made from SGneurons, as previously described (36). The pipette solution contained 135 mMK-gluconate, 5 mM KCl, 0.5 mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 5 mMHepes, and 5 mM ATP-Mg, pH 7.20, with KOH. The tip resistance of the patchpipettes was 6–12 MΩ. Series resistance was assessed from current in re-sponse to a 5-mV hyperpolarizing step. Series resistance was monitoredduring the recording session, and data were rejected if its value changed by>15%. Ionic currents were monitored with a patch-clamp amplifier, Axo-patch 700B (Molecular Devices). The data were digitized with an analog-to-digital converter (Digidata 1440A; Molecular Devices), stored on a personalcomputer using a data acquisition program (Clampex version 10; MolecularDevices), and analyzed using the software package Mini Analysis version

6.0.3 (Synaptosoft). Recordings were made in voltage-clamp mode at aholding potential of −70 mV to isolate sEPSCs. Drugs were dissolved in theKrebs solution and applied by superfusion. The dorsal root was stimulatedusing a suction electrode at a frequency of 0.2 Hz to elicit EPSCs. The Aδ- orC-afferent–mediated responses were distinguished on the basis of the con-duction velocity of the afferent fibers and stimulus thresholds. The con-duction velocity was calculated from the latency of synaptic responses andthe length of the dorsal root. The Aδ- and C-fiber–evoked responses wereconsidered monosynaptic if the latency remained constant without failurewhen the root was stimulated at 20 Hz for the Aδ-fiber–evoked EPSCs andwhen stimulated at 2 Hz for the C-fiber–evoked EPSCs (37).

Pain-Related Behavior Test. Mice were handled gently for 1 h every 48 hbefore the behavior test. Mice were injected with capsaicin (300 μM) orαβ-methylene ATP with or without A01 (total 10 μL) into the top of the hindpaw using a fine (30-G) needle filled with saline (Otsuka Pharmaceutical)containing 0.3% ethanol and 3% (vol/vol) dimethyl sulfoxide [capsaicin andA01 were dissolved in 0.3% ethanol and 3% (vol/vol) dimethyl sulfoxide,respectively]. Mice were wrapped gently in the measurer’s hand and kept ontheir backs with their snouts toward the small finger of the measurer. Miceremained very quiet during injection in this position. Their behavior wasrecorded using a digital camera (P6000; Nikon).

ACKNOWLEDGMENTS. We thank Dr. K. Shibasaki (Gunma University) fortechnical support, Dr. M. Kido (Kyushu University) for the generous gift ofthe anti-TRPV1 antibody, and Dr. U. Oh (Seoul National University) for thegenerous gift of the Ano1 cDNA. This work was supported by a Grant-in-Aidfor Scientific Research from the Ministry of Education, Culture, Sports, Sci-ence and Technology in Japan (Brain Environment 26111732) (to M.T.) andby The Salt Science Research Foundation (M.T.).

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